Affiliation: †Division of Pharmaceutics, College of Pharmacy, The University of Texas at Austin, Austin, Texas 78712, United States.

ABSTRACTThe severity and longevity of the current Ebola outbreak highlight the need for a fast-acting yet long-lasting vaccine for at-risk populations (medical personnel and rural villagers) where repeated prime-boost regimens are not feasible. While recombinant adenovirus (rAd)-based vaccines have conferred full protection against multiple strains of Ebola after a single immunization, their efficacy is impaired by pre-existing immunity (PEI) to adenovirus. To address this important issue, a panel of formulations was evaluated by an in vitro assay for their ability to protect rAd from neutralization. An amphiphilic polymer (F16, FW ∼39,000) significantly improved transgene expression in the presence of anti-Ad neutralizing antibodies (NAB) at concentrations of 5 times the 50% neutralizing dose (ND50). In vivo performance of rAd in F16 was compared with unformulated virus, virus modified with poly(ethylene) glycol (PEG), and virus incorporated into poly(lactic-co-glycolic) acid (PLGA) polymeric beads. Histochemical analysis of lung tissue revealed that F16 promoted strong levels of transgene expression in naive mice and those that were exposed to adenovirus in the nasal cavity 28 days prior to immunization. Multiparameter flow cytometry revealed that F16 induced significantly more polyfunctional antigen-specific CD8+ T cells simultaneously producing IFN-γ, IL-2, and TNF-α than other test formulations. These effects were not compromised by PEI. Data from formulations that provided partial protection from challenge consistently identified specific immunological requirements necessary for protection. This approach may be useful for development of formulations for other vaccine platforms that also employ ubiquitous pathogens as carriers like the influenza virus.

fig7: FormulationF16 improves quantitative and qualitative Ebola glycoprotein-specificCD8+ T cell responses in mice with prior exposureto adenovirus. PEI to adenovirus 5 was induced by instilling 5 ×1010 virus particles of AdNull, an E1/E3 deleted virusthat does not contain a transgene cassette, in the nasal cavity ofmice 28 days prior to immunization. (A) The systemic effector CD8+ T cell response. Ten days after vaccination,mononuclear cells from the spleen were harvested and stimulated withan Ebola GP-specific peptide and responsive cells were quantitatedby ELISpot. (B) The mucosal effector CD8+ Tcell response. Ten days after vaccination, mononuclear cells collectedfrom BAL fluid were harvested, pooled according to treatment, andstimulated with an Ebola GP-specific peptide and responsive cellswere quantitated by ELISpot. (C) The polyfunctional CD8+ T cell response. Ten days after immunization, splenocytesfrom 5 mice per treatment group were pooled and stimulated with anEbola glycoprotein-specific peptide. Each positively responding cellwas assigned to one of 7 possible combinations of IFN-γ, IL-2,and TNF-α production and quantitated as shown in the bar graph.The most potent responders, those producing all 3 cytokines in responseto stimulation, are depicted by the red arcs in the pie charts. Theproportion of cells in samples from each treatment group that produceIFN-γ is depicted by the blue arc. The number in each pie chartdenotes the percentage of triple producers found in samples from agiven treatment group. Data reflect average values ± the standarderror of the mean for six mice per group. * indicates a significantdifference with respect to the naive/unformulated group, *p < 0.05, **p < 0.01, one-way ANOVA,Bonferroni/Dunn post hoc analysis.

Mentions:
Because prior formulation candidatesdid not fully confer protection in mice in which PEI was establishedthrough the nasal mucosa, evaluation of the F16 formulation in vivo focused solely on the ability of this formulationto improve the immune response to the encoded Ebola glycoprotein underthese specific conditions. As seen in prior studies, PEI significantlycompromised the production of GP-specific IFN-γ-secreting mononuclearcells isolated from spleen (Figure 7A) andBAL fluid (Figure 7B) in animals given unformulatedvaccine (p < 0.01). PEI induced by the mucosalroute also significantly reduced the frequency of GP-specific multifunctionalCD8+ T cells elicited by the unformulated vaccine (naive,64.9 ± 4.88%, vs IN PEI/unformulated, 48.6 ± 3.66%, p < 0.05; Figure 7C).

fig7: FormulationF16 improves quantitative and qualitative Ebola glycoprotein-specificCD8+ T cell responses in mice with prior exposureto adenovirus. PEI to adenovirus 5 was induced by instilling 5 ×1010 virus particles of AdNull, an E1/E3 deleted virusthat does not contain a transgene cassette, in the nasal cavity ofmice 28 days prior to immunization. (A) The systemic effector CD8+ T cell response. Ten days after vaccination,mononuclear cells from the spleen were harvested and stimulated withan Ebola GP-specific peptide and responsive cells were quantitatedby ELISpot. (B) The mucosal effector CD8+ Tcell response. Ten days after vaccination, mononuclear cells collectedfrom BAL fluid were harvested, pooled according to treatment, andstimulated with an Ebola GP-specific peptide and responsive cellswere quantitated by ELISpot. (C) The polyfunctional CD8+ T cell response. Ten days after immunization, splenocytesfrom 5 mice per treatment group were pooled and stimulated with anEbola glycoprotein-specific peptide. Each positively responding cellwas assigned to one of 7 possible combinations of IFN-γ, IL-2,and TNF-α production and quantitated as shown in the bar graph.The most potent responders, those producing all 3 cytokines in responseto stimulation, are depicted by the red arcs in the pie charts. Theproportion of cells in samples from each treatment group that produceIFN-γ is depicted by the blue arc. The number in each pie chartdenotes the percentage of triple producers found in samples from agiven treatment group. Data reflect average values ± the standarderror of the mean for six mice per group. * indicates a significantdifference with respect to the naive/unformulated group, *p < 0.05, **p < 0.01, one-way ANOVA,Bonferroni/Dunn post hoc analysis.

Mentions:
Because prior formulation candidatesdid not fully confer protection in mice in which PEI was establishedthrough the nasal mucosa, evaluation of the F16 formulation in vivo focused solely on the ability of this formulationto improve the immune response to the encoded Ebola glycoprotein underthese specific conditions. As seen in prior studies, PEI significantlycompromised the production of GP-specific IFN-γ-secreting mononuclearcells isolated from spleen (Figure 7A) andBAL fluid (Figure 7B) in animals given unformulatedvaccine (p < 0.01). PEI induced by the mucosalroute also significantly reduced the frequency of GP-specific multifunctionalCD8+ T cells elicited by the unformulated vaccine (naive,64.9 ± 4.88%, vs IN PEI/unformulated, 48.6 ± 3.66%, p < 0.05; Figure 7C).

Affiliation:
†Division of Pharmaceutics, College of Pharmacy, The University of Texas at Austin, Austin, Texas 78712, United States.

ABSTRACTThe severity and longevity of the current Ebola outbreak highlight the need for a fast-acting yet long-lasting vaccine for at-risk populations (medical personnel and rural villagers) where repeated prime-boost regimens are not feasible. While recombinant adenovirus (rAd)-based vaccines have conferred full protection against multiple strains of Ebola after a single immunization, their efficacy is impaired by pre-existing immunity (PEI) to adenovirus. To address this important issue, a panel of formulations was evaluated by an in vitro assay for their ability to protect rAd from neutralization. An amphiphilic polymer (F16, FW ∼39,000) significantly improved transgene expression in the presence of anti-Ad neutralizing antibodies (NAB) at concentrations of 5 times the 50% neutralizing dose (ND50). In vivo performance of rAd in F16 was compared with unformulated virus, virus modified with poly(ethylene) glycol (PEG), and virus incorporated into poly(lactic-co-glycolic) acid (PLGA) polymeric beads. Histochemical analysis of lung tissue revealed that F16 promoted strong levels of transgene expression in naive mice and those that were exposed to adenovirus in the nasal cavity 28 days prior to immunization. Multiparameter flow cytometry revealed that F16 induced significantly more polyfunctional antigen-specific CD8+ T cells simultaneously producing IFN-γ, IL-2, and TNF-α than other test formulations. These effects were not compromised by PEI. Data from formulations that provided partial protection from challenge consistently identified specific immunological requirements necessary for protection. This approach may be useful for development of formulations for other vaccine platforms that also employ ubiquitous pathogens as carriers like the influenza virus.